The absence of adequate numbers of hemostatically active blood platelets is associated with many disease states, some of which can only be treated by transfusion of blood products containing large numbers of viable platelets. Freshly obtained blood platelets mediate hemostasis by converting, where properly instructed, from discs to spiny pleated spheres that attach to breaks in blood vessels and to other platelets. This process, referred to as platelet activation, is triggered by a variety of different agonists, including thrombin, adenosine diphosphate (ADP), thromboxanes, collagen, von Willebrand's factor, as well as upon contact of platelets with glass.
Current practice permits platelets to be stored no longer than several days, after which the platelets are no longer hemostatically active and are discarded as "outdated". It is estimated that about 15% of procured units of blood are discarded as outdated. As a result of the short platelet shelf life, a large supply of donated blood is required to sustain each patient requiring platelet replacement therapy.
Given the problems of platelet availability, various attempts have been made to preserve platelets for longer periods of time with retention of hemostatic activity. Most of this work was done in the 1960's and early 1970's and culminated in the practice of room temperature storage. These studies revealed that while room temperature storage led rapidly to significant reduction in hemostatic function, the phenomenon of cold-induced platelet activation had more deleterious effects (Murphy, P. H. and Gardener, F. H., 1969 N. Engl. J. Med. 280:1094-1098; Handin, R. I. and Valeri, C. R., 1973 J. Engl. J. Med. 285:538-543). More recently, research has focused on the modification of platelet storage packs or bags to increase porosity and gas exchange, on nutrients, metabolites, pH and protease inhibitors (e.g., Murphy, S. et al., 1982 Blood 60:194-200; Rinder, H. M. and Snyder, E. L., 1992 Blood Cells 18:445-456). Because storage at non-refrigerated temperatures has been associated with microbial contamination of transfused platelets (Bennett, J. V., 1971 N. Engl. J. Med. 285:457-458; Buckholz, D. H., et al., 1971 N. Engl. J. Med. 285:429-433; Morrow, J. F., et al., 1991 JAMA 266:555-558) the Food and Drug Administration (FDA) limits platelet storage to five days.
To date, efforts to store platelets at reduced temperatures have proven unsuccessful because of the morphological changes which platelets undergo in response to cold temperatures. These changes, collectively referred to as "cold-induced platelet activation", result in substantially impaired hemostatic function. In contrast to freshly obtained platelets, platelets that have been rewarmed following cold-induced activation share many structural features with glass-activated platelets but have substantially impaired hemostatic activity. Thus, although (agonist- or glass-induced) platelet activation and cold-induced platelet activation have in common some structural similarities, these activation processes yield quite distinctive functional results. To understand the processes which comprise agonist-and/or cold-induced activation and the differences between the two types of activation, the cytoskeletal structure of the resting platelet must first be considered.
Prior to activation, the resting platelet contains a highly organized cytoskeletal structure, with actin representing about a fifth of the total protein (Hartwig, J., 1992 J. Cell Biology 118(6):1421-1442). About half of the actin in resting platelets is present as actin monomer ("G-actin") and is stored as a 1:1 complex with beta4-thymosin or profilin. The remainder of the actin in resting platelets is organized into long filaments ("F-actin") which radiate outwardly from the platelet center. The filaments have a fast-growing end, the "barbed end", to which the actin monomers are added in a process alternatively referred to as actin assembly or actin polymerization.
Spontaneous actin assembly from monomers in vitro proceeds through a thermodynamically unfavorable nucleation step that limits the initial rate of this polymerization reaction. In vivo, various proteins regulate platelet activation by association with actin monomers and/or filaments. The presence in platelets of nearly stoichiometric quantities of actin monomer binding proteins, e.g. profilin and beta 4-thymosin, with affinities for actin monomer in the micromolar range, presumably prevents spontaneous nucleation in vivo (Safer, D., et al., 1991 J. Biol. Chem. 266:4029-4032; Weeds, A. G., et al., 1992 Biochem. Soc. Trans. 19:1016-1020). By associating with actin monomers, these "sequestering proteins" render the monomers incapable of adding to the free pointed ends of actin filaments and less capable of adding to the (uncapped) barbed ends of actin filaments.
The exact interplay of these regulatory proteins with actin monomers and filaments and their involvement in platelet activation is not precisely understood. In the resting platelet, actin filaments bind via actin-binding proteins ("abp") to a dense spectrin-rich shell that laminates the plasma membrane (see e.g., Hartwig, J. and DeSisto, M., 1991 J. Cell Biol. 112:407-425). We have observed that upon stimulation by an agonist, such as thrombin, the resting platelet swells, presumably as a result of actin filament severing (see Hartwig, J., 1992 supra.). It is known that severing requires an increase in the intracellular free calcium concentration (Hartwig, J. and Yin, H. L., 1987 BioEssays 7:176-179).
Exposure of platelets to thrombin increases the intracellular calcium concentration to near micromolar levels in the absence of external calcium and to greater than micromolar levels when calcium is a component of the surrounding medium (see e.g., Oda, A., et al., 1991 Am. J. Physiol. 260:C242-C248). Calcium at micromolar levels leads to the formation of gelsolin-actin complexes in vitro (Stossel, T., 1989 J. Biol. Chem. 264:18261-18264). In the resting platelet, &gt;95% of the gelsolin is free, i.e., not complexed to actin (Lind, et al., 1987 J. Cell Biol. 105:833-842). Free gelsolin (not gelsolin-actin complexes) reportedly plays a role in calcium-dependent actin filament severing (Janmey, P. A., et al., 1985 Biochemistry 24:3714-3723). Loading cells with permeant calcium chelators reportedly quenches the increase in intracellular calcium concentration in response to agonists such as thrombin (Davies, T. D., et al., 1989 J. Biol. Chem. 264:19600-19606).
Various intracellular calcium chelating agents have been used as research tools to elucidate the role of calcium in platelet activation. These include derivatives and analogues of the calcium chelator BAPTA developed by Tsien et al., (see e.g., U.S. Pat. No. 4,603,209). Many of these chelators exhibit an increase in fluorescence emission (in response to appropriate excitation) upon binding free calcium. However, to be useful as intracellular chelating agents, these calcium chelators had to be derivatized with lipophilic groups, i.e., to render the chelators capable of penetrating the platelet membrane and entering the cytosol. Such intracellular calcium chelators have been used to measure intracellular calcium concentrations in human blood platelets at rest and during activation (Cobbold, P. and Rink, T., 1987 Biochem. J. 248:313-328). Very low intracellular calcium concentrations were achieved when large amounts of the chelators were loaded into the cytosol in the absence of an exogenous source of free (unchelated) calcium (Cobbold and Rink, 1987, supra.).
Platelet activation is manifested by transformation of the platelet into a compact sphere from which extend spines (filopodia) and veils ("lamellipodial networks") (FIG. 1). The filopodia comprise bundles of actin filaments ("filopodial bundles"). The veils contain shorter actin filaments and represent a second type of filament organization. The generation of both of these actin structures requires gelsolin. We believe that removal of gelsolin from the core actin network, i.e., the population of actin filaments deep within the platelet, leads to formation of the filopodial bundles and that removal of gelsolin from severed actin filaments leads to formation of the lamellipodial network.
Much of what is known about the structural changes accompanying platelet activation has been learned from studying the barbed end actin polymerization activity of detergent-demembranated platelets in various states of activation. Barbed end actin polymerization activity is determined by observing the rate at which newly added actin monomer is incorporated into platelet filaments (see e.g., Hartwig, J. and Janmey, P., 1989 Biochim. Biophys. Acta. 3030:64-71). Because cytochalasin B is a well known inhibitor of actin assembly onto the barbed ends of actin filaments, the existence and extent of barbed end activity is determined by observing the effect of cytochalasin B on the rate at which actin monomers are added to the barbed ends of actin filaments.
The cytochalasins and the related chaetoglobosins constitute a class of more than 24 structurally and functionally related mold metabolites. Several publications have reported that cytochalasin B prevents some of the platelet shape changes associated with cold-induced activation, but that other changes, e.g., distortions of intracellular membranes, were not prevented (White, J. B. and Krivit, W., 1967 Blood 30:625; White, J. G., 1982 Am. J. Path. 108:184). More recently, the cytochalasins have been reported to alter actin-based cytoskeletal morphology (see e.g., Schliwa, M., 1982 J. Cell Biol. 92:79-91) and inhibit actin polymerization (see e.g., Mooseker, M. S., 1986 J. Cell Biol. 102:282-288).
In vitro studies using purified actin indicate that cytochalasins bind to the barbed end of actin filaments and inhibit its polymerization (see e.g., Lin et al., 1980 J. Cell Biol. 84:455-460) by reducing the rate of monomer addition to the barbed end of growing filaments (see e.g., Ohmori, H., et al., 1992, J. Cell Biol. 116(4):933-941 and references cited therein). Although the detailed mechanism by which the cytochalasins inhibit actin polymerization has not been elucidated (e.g., Cooper, J. A., 1987 J. Cell Biol. 105:1473-1478), it is believed that the cytochalasins and related compounds interfere with the dynamic equilibrium that exists in nonmuscle cells between actin filaments (F-actin) and monomeric actin (G-actin) (see e.g., Spector, I., et al., 1989 Cell Motility and the Cytoskeleton 13:127-144 and references cited therein).
The above-cited references disclose the use of agents such as cytochalasin B and intracellular calcium chelators for characterizing the biochemical and morphological changes that occur during agonist-and/or glass-induced platelet activation. However, none of the cited references disclose the use of such agents, alone or in combination, for modulating or preventing cold-induced platelet activation. Accordingly, there is still a need for methods and pharmaceutical compositions to preserve platelets. In particular, there is still a need for methods for preserving platelets at cryopreservation temperatures, which methods prevent cold-induced platelet activation. Such methods would permit the preservation of blood platelets with preserved hemostatic activity for longer periods of time than are currently possible.